Two-Photon Microperimetry: A Media Opacity-Independent Retinal Function Assay

Abstract

Purpose: Compare results obtained using infrared two-photon microperimetry (2PM-IR) with conventional visual function tests in healthy subjects of varying ages with and without simulated media opacities.

Methods: Subjects from two separate cohort studies completed cone contrast threshold (CCT) testing, conventional microperimetry, visible light microperimetry from a novel device (2PM-Vis), and infrared two-photon microperimetry. The first cohort study, which consisted of six healthy volunteers (23 to 29 years of age), evaluated the effects of simulated media opacities on visual performance testing. Subjects underwent testing on four visual function devices nine separate times under the following conditions: no filter, red filter, green filter, blue filter, light brown filter, dark brown filter, polarized black filter (0° rotation), and polarized black filter (90° rotation). Subjects subsequently performed 2PM-IR and 2PM-Vis testing without a filter in the mydriatic state. The second cohort study evaluated the effect of age on visual test performance in 42 healthy subjects split between two groups (ages 20-40 years and 60-80 years).

Results: Retinal sensitivity measured by 2PM-IR demonstrated lower variability than all other devices relying on visible spectrum stimuli. Retinal sensitivity decreased proportionally with the transmittance of light through each filter. CCT scores and retinal sensitivity decreased with age in all testing modalities. Visible spectrum testing modalities demonstrated larger test result differences between young and old patient cohorts; this difference was inversely proportional to the wavelength of the visual function test.

Conclusions: 2PM-IR mitigates media opacities that may mask small differences in retinal sensitivity when tested with conventional visual function testing devices.

Translational relevance: Conventional visual function tests that emit visible light may not detect differences in retinal function during the early stages of age-related diseases due to the confounding effects of cataracts. Infrared light, which has greater transmittance through ocular tissue, may reliably quantify retinal sensitivity and thereby detect degenerative changes early on.

Figure 1.

(A) 2PM is capable of testing retinal sensitivity using single-photon visible (2PM-Vis) and two-photon infrared (2PM-IR) stimuli. (B) Configuration for measuring cone contrast/retinal sensitivity under the nine different testing conditions.

Figure 2.

Spectral transmission of filters (lines) superimposed on the spectral emission of RGB light from the CCT monitor (shaded regions). Horizontal bars above the plot represent opsin spectral absorbances, with vertical lines highlighting peak spectral absorption. Vertical dashed lines indicate wavelengths of visible green 2PM-Vis (523 nm) and pulsed infrared 2PM-IR (1045 nm) stimulation.

Figure 3.

Effect of filters on visual function testing. (A) Foveal microperimetry with 2PM-IR, 2PM-Vis, and cMP testing. (B) Cone contrast threshold testing for each opsin.

Figure 4.

Linear regression of averaged retinal sensitivities versus filter spectral transmission for (A) 2PM-IR (R² = 0.965, β = –62.967, P < 0.001) and (B) 2PM-Vis (R² = 0.637, β = 11.002, P = 0.005).

Figure 5.

Color vision score distributions and means (95% CIs) by age. CCT scores and retinal sensitivity were compared between young (20–40 years of age) and old (60–80 years of age) healthy subjects using conventional and novel visual function testing devices. Asterisks indicate statistically significant differences (P < 0.05) between the young and old groups. Slope indicates decrease in score per year increase in age for each test, modeled using linear regression.